RESUMO
How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos.
Assuntos
Blastocisto , Cromatina/metabolismo , Animais , Sítios de Ligação , Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Fator de Ligação a CCAAT/metabolismo , Mapeamento Cromossômico , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras GenéticasRESUMO
To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using â¼100 cells and single cells, respectively. liMAB-seq analysis of preimplantation embryos reveals the oxidation of 5mC to 5fC/5caC and the positive correlation between chromatin accessibility and processivity of ten-eleven translocation (TET) enzymes. scMAB-seq captures the cell-to-cell heterogeneity of 5fC and 5caC and reveals the strand-biased distribution of 5fC and 5caC. scMAB-seq also allows the simultaneous high-resolution mapping of sister chromatid exchange (SCE), facilitating the study of this type of genomic rearrangement. Therefore, our study not only establishes new methods for the genomic mapping of active DNA demethylation using limited numbers of cells or single cells but also demonstrates the utilities of the methods in different biological contexts.
Assuntos
Mapeamento Cromossômico/métodos , Metilação de DNA , Genômica/métodos , Análise de Célula Única/métodos , Troca de Cromátide Irmã , Animais , Blastômeros/metabolismo , Replicação do DNA , Embrião de Mamíferos , CamundongosRESUMO
Sulforaphane (SFaN) is a food-derived compound with several bioactive properties, including atherosclerosis, diabetes, and obesity treatment. However, the mechanisms by which SFaN exerts its various effects are still unclear. To elucidate the mechanisms of the various effects of SFaN, we explored novel SFaN-binding proteins using SFaN beads and identified acyl protein thioesterase 2 (APT2). We also found that SFaN binds to the APT2 via C56 residue and attenuates the palmitoylation of APT2, thereby reducing plasma membrane localization of APT2. This study reveals a novel bioactivity of SFaN as a regulator of APT2 protein palmitoylation.
Assuntos
Isotiocianatos , Lipoilação , Sulfóxidos , Tioléster Hidrolases , Isotiocianatos/metabolismo , Isotiocianatos/farmacologia , Isotiocianatos/química , Sulfóxidos/farmacologia , Sulfóxidos/metabolismo , Sulfóxidos/química , Humanos , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/química , Lipoilação/efeitos dos fármacos , Ligação Proteica , Células HEK293 , Membrana Celular/metabolismoRESUMO
Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we showed that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of 2 hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and 1 carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in the livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fator 4 Nuclear de Hepatócito , Fígado , Regiões Promotoras Genéticas , Animais , Humanos , Masculino , Camundongos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Camundongos Knockout , Elementos de Resposta , Ativação Transcricional , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashions. After fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci, including imprinting control regions. How parental chromatin becomes equalized and how imprinting control regions escape from this reprogramming is largely unknown. Here we profile parental allele-specific DNase I hypersensitive sites in mouse zygotes and morula embryos, and investigate the epigenetic mechanisms underlying these allelic sites. Integrated analyses of DNA methylome and tri-methylation at lysine 27 of histone H3 (H3K27me3) chromatin immunoprecipitation followed by sequencing identify 76 genes with paternal allele-specific DNase I hypersensitive sites that are devoid of DNA methylation but harbour maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induces maternal allele expression. H3K27me3-dependent imprinting is largely lost in the embryonic cell lineage, but at least five genes maintain their imprinted expression in the extra-embryonic cell lineage. The five genes include all paternally expressed autosomal imprinted genes previously demonstrated to be independent of oocyte DNA methylation. Thus, our study identifies maternal H3K27me3 as a DNA methylation-independent imprinting mechanism.
Assuntos
Metilação de DNA , Impressão Genômica , Histonas/metabolismo , Alelos , Animais , Blastocisto/metabolismo , Linhagem da Célula , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica , Histonas/química , Lisina/metabolismo , Masculino , Camundongos , Mórula/metabolismo , Oócitos/metabolismo , Zigoto/citologia , Zigoto/metabolismoRESUMO
Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein. A loss of TSC2 function induces hyperactivation of mechanistic target of rapamycin (mTOR). The C-terminal region of TSC2 contains a calmodulin (CaM) binding region and the CaM-TSC2 interaction contributes to proper mTOR activity. However, other downstream signaling pathways/effectors activated by the CaM-TSC2 complex have not been fully elucidated. In this study, we found that activation of Ca2+/CaM signaling resulted in the translocation of membrane-associated TSC2 to the nucleus and suppressed the transcriptional activity of the vitamin D receptor (VDR). TSC2 was released from the membrane in an activated CaM-dependent state in rat brain and HeLa cells. It subsequently formed a transcriptional complex to partially suppress the transcription of CYP24A1, a well-known VDR target gene. These data suggest, in part, that TSC2 attenuates VDR-associated transcriptional regulation via Ca2+/CaM signaling.
Assuntos
Calmodulina , Esclerose Tuberosa , Ratos , Humanos , Animais , Calmodulina/metabolismo , Vitamina D3 24-Hidroxilase/metabolismo , Cálcio/metabolismo , Células HeLa , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein that is partially regulated by insulin, energy, oxygen, and growth factors. Mutations in the TSC2 gene and loss of TSC2 promote cell growth by the mammalian target of rapamycin complex 1 (mTORC1) activation. Furthermore, S-adenosylmethionine (SAM) sensor upstream of mTORC1 indirectly inhibits mTORC1 activity via the methionine metabolite SAM. Here, we investigated the effects of methionine on insulin/TSC2/mTORC1 activity. Our results showed that methionine affected TSC2 stability and abolished TSC2 localization to the lysosome. Moreover, activation of insulin signaling contributed to TSC2 degradation in a methionine deprivation-dependent manner. Thus, methionine and insulin crosstalk occurred via TSC2.
Assuntos
Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/química , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Células HEK293 , Células HeLa , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Fosforilação , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
ß-catenin is a multi-functional protein with a central role in regulating embryonic development and tissue homeostasis. The abnormal accumulation of ß-catenin, due to disrupted ß-catenin degradation or unregulated ß-catenin synthesis, causes the development of cancer. A recent study showed that the overexpression of proto-oncogene serine/arginine-rich splicing factor 9 (SRSF9) promotes ß-catenin accumulation via binding ß-catenin mRNA and enhancing its translation in a manner that is dependent on the mechanistic target of rapamycin (mTOR). However, the regulation of the interaction between SRSF9 and mRNA of ß-catenin remains unclear. Here, we show that AMP-activated protein kinase (AMPK) phosphorylates SRSF9 at the RNA-interacting SWQDLKD motif that plays a major role in determining substrate specificity. The phosphorylation by AMPK inhibits the binding of SRSF9 to ß-catenin mRNA and suppresses ß-catenin protein synthesis caused by SRSF9 overexpression without changing the ß-catenin mRNA levels. Our findings suggest that AMPK activators are potential therapeutic targets for SRSF9-derived overproduction of ß-catenin in cancer cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , beta Catenina/biossíntese , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Técnicas In Vitro , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Proto-Oncogene Mas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Processamento de Serina-Arginina/química , Fatores de Processamento de Serina-Arginina/genética , Especificidade por Substrato , beta Catenina/genéticaRESUMO
Liver X receptors (LXR) α and ß are a family of nuclear receptors that regulate lipogenesis by controlling the expression of the genes involved in the synthesis of fatty acids. MID1IP1, which encodes MIG12, is a target gene of LXR. MIG12 induces fatty acid synthesis by stimulating the polymerization-mediated activation of acetyl-CoA carboxylase (ACC). Here, we show that LXR's activation stimulates ACC polymerization in HepG2 cells by increasing the expression of MIG12. A knockdown of MID1IP1 abrogated the stimulation completely. The mutations of MIG12's leucine-zipper domain reduced the interaction between MIG12 and ACC, thus decreasing the MIG12's capacity to stimulate ACC polymerization. These results indicate that LXR's activation stimulates lipogenesis not only through the induction of the genes encoding lipogenic enzymes but also through MIG12's stimulation of ACC polymerization.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Receptores X do Fígado/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Lipogênese , PolimerizaçãoRESUMO
BACKGROUND: White rice and its unrefined form, brown rice, contain numerous compounds that are beneficial to human health. However, the starch content of rice can contribute to obesity, a main risk factor for nonalcoholic fatty liver disease (NAFLD). OBJECTIVES: We investigated the effect of rice consumption on NAFLD and its underlying molecular mechanism. METHODS: We randomly divided 7-week-old male obese Zucker (fa/fa) rats, an animal model of NAFLD, into 3 groups (n = 10 each) fed 1 of 3 diets for 10 weeks: a control diet (Cont; AIN-93G diet; 53% cornstarch), a white rice diet (WR; AIN-93G diet with cornstarch replaced with white rice powder), or a brown rice diet (BR; AIN-93G diet with cornstarch replaced with brown rice powder). Liver fat accumulation and gene expression related to lipid and vitamin A metabolisms, including retinoic acid (RA) signaling, were analyzed. RESULTS: Hepatic lipid values were significantly decreased in the BR group compared with the Cont group, by 0.4-fold (P < 0.05). The expression of genes related to hepatic fatty acid oxidation, such as carnitine palmitoyltransferase 2, was approximately 2.1-fold higher in the BR group than the Cont group (P < 0.05). The expression of peroxisomal acyl-coenzyme A oxidase 1 and acyl-CoA dehydrogenase medium chain was also significantly increased, by 1.6-fold, in the BR group compared with the Cont group (P < 0.05). The expression of VLDL-secretion-related genes, such as microsomal triglyceride transfer protein, was also significantly higher in the BR group (2.4-fold; P < 0.05). Furthermore, aldehyde dehydrogenase 1 family member A1, an RA synthase gene, was 2-fold higher in the BR group than the Cont group (P < 0.05). CONCLUSIONS: Brown rice prevented development of NAFLD in obese Zucker (fa/fa) rats. The beneficial effects of pregelatinized rice on NAFLD could be manifested as increased fatty acid oxidation and VLDL secretion, which are regulated by RA signaling.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Oryza , Animais , Metabolismo dos Lipídeos , Lipídeos , Fígado/metabolismo , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Ratos , Ratos Zucker , Tretinoína/metabolismoRESUMO
AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK ß regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic processes and activating catabolic processes. Recent studies have demonstrated that metformin, which is an AMPK activator, modifies alternative precursor mRNA (pre-mRNA) splicing. However, no direct substrate of AMPK for alternative pre-mRNA splicing has been reported. In the present study, we identified the splicing factor serine/arginine-rich splicing factor 1 (SRSF1) as a novel AMPK substrate. AMPK directly phosphorylated SRSF1 at Ser133 in an RNA recognition motif. Ser133 phosphorylation suppressed the interaction between SRSF1 and specific RNA sequences without altering the subcellular localization of SRSF1. Moreover, AMPK regulated the SRSF1-mediated alternative pre-mRNA splicing of Ron, which is a macrophage-stimulating protein receptor, by suppressing its interaction with exon 12 of Ron pre-mRNA. The findings of this study revealed that the AMPK-dependent phosphorylation of SRSF1 at Ser133 inhibited the ability of SRSF1 to bind RNA and regulated alternative pre-mRNA splicing.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Processamento Alternativo , Éxons , Precursores de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Células HEK293 , Humanos , Células MCF-7 , Fosforilação , Precursores de RNA/genética , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.
RESUMO
AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by suppressing anabolic processes and activating catabolic processes. AMPK activators are an important therapeutic target for metabolic syndrome due to favorable physiological effects of AMPK activation on metabolism. Recent studies show that niclosamide, an FDA-approved anthelmintic drug that exerts an uncoupling effect on the mitochondria of the parasite, improves blood glucose levels and reduces hepatic steatosis in mice via AMPK activation. Niclosamide is thought to activate AMPK by increasing AMP/ATP ratio through mitochondrial uncoupling, but details of its action remain unclear. In this study, we found that niclosamide also activates the AMPK complex, which contains the AMP-insensitive γ subunit. Further, niclosamide shows greater AMPK activation for the AMPK complex containing ß2 subunit, but not the ß1 subunit. This effect was inhibited by substituting the Ser108 residue of the ß2 subunit to alanine. Niclosamide displays a novel AMPK activation mechanism independent of the increase in AMP/ATP ratio.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Anti-Helmínticos/farmacologia , Niclosamida/farmacologia , Proteínas Quinases Ativadas por AMP/química , Monofosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Treonina/metabolismoRESUMO
Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of genes involved in fatty acid and cholesterol biosynthetic pathways. The present study showed that the flavonoid chrysin impairs the fatty acid synthase promoter. Chrysin reduces the expression of SREBP target genes, such as fatty acid synthase, in human hepatoma Huh-7 cells and impairs de novo synthesis of fatty acids and cholesterol. Moreover, it reduces the endogenous mature, transcriptionally active forms of SREBPs, which are generated by the proteolytic processing of precursor forms. In addition, chrysin reduces the enforced expressing mature forms of SREBPs and their transcriptional activity. The ubiquitin-proteasome system is not involved in the chrysin-mediated reduction of SREBPs mature forms. These results suggest that chrysin suppresses SREBP activity, at least partially, via the degradation of SREBPs mature forms. Abbreviations: ACC1: acetyl-CoA carboxylase 1; DMEM: Dulbecco's modified Eagle's medium; FAS: fatty acid synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 25-HC: 25-hydroxycholesterol; HMGCS: HMG-CoA synthase; LDH: lactate dehydrogenase; LPDS: lipoprotein-deficient serum; PI3K: phosphatidylinositol 3-kinase; SCD1: stearoyl-CoA desaturase; SREBPs: sterol regulatory element-binding proteins.
Assuntos
Flavonoides/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Linhagem Celular Tumoral , Colesterol/biossíntese , Ácido Graxo Sintases/genética , Ácidos Graxos/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Proteólise , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Proteínas de Ligação a Elemento Regulador de Esterol/genéticaRESUMO
Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Interleucinas/farmacologia , Prurido/induzido quimicamente , Receptores de Interleucina/metabolismo , Células A549 , Animais , Células CHO , Linhagem Celular , Cricetulus , DNA Complementar/metabolismo , Humanos , Cinética , Macaca fascicularis , Masculino , Camundongos , Prurido/metabolismo , Transdução de Sinais , Pele/imunologia , Pele/patologia , Dermatopatias/imunologia , Dermatopatias/patologiaRESUMO
Tuberous sclerosis complex (TSC) presents as benign tumors that affect the brain, kidneys, lungs and skin. The inactivation of TSC2 gene, through loss of heterozygosity is responsible for tumor development in TSC. Since TSC patients are carriers of heterozygous a TSC2; mutation, to reveal the risk factors which these patients carry prior to tumor development is important. In this experiment, Eker rat which carry a mutation in this TSC2 gene were analyzed for their metabolic changes. Wild-type (TSC2+/+) and heterozygous mutant TSC2 (TSC2+/-) Eker rats were raised for 100 days. As a result, the Eker rats were found to exhibit hyperglycemia and hyperketonemia. However the high ketone body production in the liver was observed without accompanying increased levels of plasma free fatty acids or insulin. Further, production of the ketone body ß-hydroxybutyrate was inhibited due to the low NADH/NAD(+) ratio resulting from the restraint on glycolysis, which was followed by inhibition of the malate-aspartate shuttle and TCA cycle. Therefore, we conclude that glycolysis is restrained in the livers of TSC2 heterozygous mutant rats, and these defects lead to abnormal production of acetoacetate.
Assuntos
Glicemia/metabolismo , Hiperglicemia/metabolismo , Cetose/metabolismo , Fígado/metabolismo , Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Glicólise , Hiperglicemia/complicações , Corpos Cetônicos/biossíntese , Masculino , Ratos , Ratos Long-Evans , Ratos Transgênicos , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Vitamin B12 deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B12 deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B12 deficiency suggests that vitamin B12 plays an important role in Pi homeostasis.
Assuntos
Reabsorção Óssea , Rim/metabolismo , Osteoclastos/patologia , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/fisiologia , Deficiência de Vitamina B 12/fisiopatologia , Animais , Homeostase , RatosRESUMO
Tuberous sclerosis complex 2 (TSC2) is a mediator of insulin signal transduction, and a loss of function in TSC2 induces hyperactivation of mTORC1 pathway, which leads to tumorigenesis. We have previously demonstrated that Eker rat model, which is heterozygous for a TSC2 mutation, exhibits hyperglycemia and hyperketonemia. The present study was to investigate whether these changes also can affect metabolism in skeletal muscle of the Eker rat. Wild-type (TSC2+/+) and Eker (TSC2+/-) rats underwent an oral glucose tolerance test, and the latter showed decrease in whole-body glucose utilization. Additionally, reductions in the expression of glycolysis-, lipolysis-, and ketone body-related genes in skeletal muscle were observed in Eker rats. Furthermore, ATP content and mitochondrial DNA copy number were lower in skeletal muscle of Eker rats. These data demonstrate that heterozygous to mutation TSC2 not only affects the liver metabolism, but also skeletal muscle metabolism, via mitochondrial dysfunction.
Assuntos
Carcinoma de Células Renais/genética , Hiperglicemia/genética , Insulina/metabolismo , Neoplasias Renais/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Teste de Tolerância a Glucose , Heterozigoto , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Insulina/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Mitocôndrias/patologia , Músculo Esquelético/patologia , Ratos , Ratos Long-Evans , Transdução de Sinais , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiênciaRESUMO
Megalin is a scavenger receptor that serves in the endocytosis of a highly diverse group of ligands that includes Vitamin B12. We found an accumulation of megalin closed to apical region in renal proximal tubule cells of Vitamin B12-deficient rats. Interestingly, Vitamin B12 levels also controlled resorption of renal retinol binding protein. Using L2 yolk sac cells, megalin localized to the submembrane compartment by methylmalonic acid (MMA), which accumulates during vitamin B12 deficiency. In addition, MMA inhibited megalin-mediated endocytosis via YWTD repeats motif in an ectodomain of megalin. Therefore, megalin endocytosis may be regulated by MMA.